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5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
5-羟甲基胞嘧啶 (5-hmC)特异性富集   

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Abstract

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.

Keywords: 5-hydroxymethylcytocine(5-羟甲基胞嘧啶), Capture(采集), Sequencing(测序)

Materials and Reagents

  1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number:  S3014 )
  2. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number:  E9884 )
  3. Trizma base (Tris) (Sigma-Aldrich, catalog number:  T1503 )
  4. HEPES (GenScript USA, catalog number:  C01621 )
  5. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number:  M8266 )
  6. Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number:  D128 )
  7. Tween-20 (Sigma-Aldrich, catalog number:  P9416 )
  8. DBCO-S-S-PEG3-Biotin conjugate (Click Chemistry Tools, catalog number:  A112P3 )
  9. 1,4-Dithiothreitol, ultrapure (DTT) (Roche Applied Science, catalog number: 3117006001 )
  10. Hydroxymethyl Collector (Active Motif, catalog number: 55013 )
  11. Wizard Genomic DNA Purification Kit (Promega Corporation, catalog number:  A1120 )
  12. 10 kDa Amicon Ultra-0.5 ml Centrifugal Filters (EMD Millipore, catalog number:  UFC501008 )
  13. QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
  14. Micro Bio-Spin 6 Column (Bio-Rad Laboratories, catalog number:  732-6222 )
  15. Dynabeads MyOne Streptavidin C1 (Life Technologies, Invitrogen™, catalog number:  650-01 )
  16. Qiagen MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
  17. UltraPure Agarose (Life Technologies, Invitrogen™, catalog number:  16500500 )
  18. β-glucosyltransferase (β-GT) (New England Biolabs)
  19. 10x β-GT reaction buffer (see Recipes)
  20. 2x binding and washing (B&W) buffer (see Recipes)
  21. TE (see Recipes)

Equipment

  1. Sonication device (Covaris)
  2. Desktop centrifuge
  3. Water bath (Thermo Fisher Scientific)
  4. Gel running apparatus (Bio-Rad Corporation)
  5. Nanodrop 1000 (Thermo Fisher Scientific)
  6. Labquake tube shaker (Barnstead/Thermolyne)
  7. Magnetic separation stand (Promega Corporation, catalog number:  Z5342 )
  8. Qubit 2.0 Fluorometer (Life Technologies, Invitrogen™)

Procedure

  1. Fragment purified genomic DNA into short fragments depending on different genome-wide sequencing platforms (usually sonicated to 300 bp). Follow the manual from Covaris to set up the fragmentation program for 300 bp. DNA buffer for sonication can be TE (10 mM Tris pH 8.0, 1 mM EDTA), EB (10 mM Tris pH 8.5) or water.
  2. Verify the size distribution of the fragmented genomic DNA on 2% agarose gel using 100-bp DNA marker. Measure the DNA concentration after fragmentation using Nanodrop or by fluorometric quantification of dsDNA, ideal concentration should be higher than 600 ng/μl.
  3. Concentrate the DNA if necessary by 10 kDa Amicon Ultra-0.5 ml Centrifugal Filters or ethanol precipitation. Measure DNA concentration again after concentration using Nanodrop.
  4. Use appropriate starting DNA amounts and set up the reaction according to the following conditions (for brain tissue).

     Component Volume   Final Concentration
     Water  _ μl  
     10x β-GT reaction buffer  2 μl  1x
     up to 10 μg genomic DNA  _ μl  up to 500 ng/μl
     UDP-6-N3-Glc (3 mM)  0.67 μl  100 μM
     β-GT (40 μM)  1 μl  2 μM
     Total volume  20 μl  

  5. Mix by pipetting and incubate in a 37 °C water bath for 1 h.
  6. Clean up the reaction with QIAquick Nucleotide Removal Kit, using 10 μg DNA per column. Elute with 30 μl water per column and combine.
  7. Dissolve 10 mg DBCO-S-S-PEG3-Biotin conjugate in 384 μl DMSO to make 30 mM stock solution and store at -80 °C. Dilute stock solution with DMSO to make 4.5 mM working solution and store at -20 °C. Stock solution is stable at -80 °C for years. Working solution is stable at -20 °C for 3 months.
  8. Add DBCO-S-S-PEG3-Biotin conjugate working solution in the eluted DNA solution from step 6) to a final concentration of 150 μM (i.e. 1 μl of working solution per 30 μl DNA solution).
  9. Mix by pipetting and incubate in a 37 °C water bath for 1 h.
  10. Clean up the reaction with QIAquick Nucleotide Removal Kit, using 10 μg DNA per column. Elute with at least 30 μl water per column and combine. The ideal combined elution volume is 100 μl, maximum volume is 150 μl.
  11. Measure DNA concentration using Nanodrop, calculate the DNA recovery.
  12. Wash 50 μl of Dynabeads MyOne Streptavidin C1 three times with 100 μl of 1x B&W buffer following its manual. Separate the beads with a magnetic stand and resuspend the beads in 100 μl of 2x B&W buffer.
  13. Save 10 ng DNA from step 11), add the rest (100 to 150 μl) to the resuspended beads from the previous step.
  14. Incubate for 15 min at room temperature with gentle rotation on a Labquake tube shaker.
  15. Separate the beads with a magnetic stand and wash the beads three times with 200 μl of 1x B&W buffer.
  16. Elute the DNA by incubating the beads in 50 μl freshly prepared 100 mM DTT. for 2 h at room temperature with gentle rotation on a Labquake tube shaker.
  17. Separate the beads with a magnetic stand. Aspirate the eluent and load to a Micro Bio-Spin 6 Column following its instruction to remove the DTT.
  18. Purify the eluted DNA from previous step by Qiagen MinElute PCR Purification Kit and elute DNA in 10 μl EB buffer. Quantify DNA using Qubit Fluorometer. The DNA is ready for downstream genome-wide sequencing library preparation.

Recipes

  1. 10x β-GT reaction buffer
    500 mM HEPES (pH 7.9)
    250 mM MgCl2
  2. 2x binding and washing (B&W) buffer
    10 mM Tris (pH 7.5)
    1 mM EDTA
    2 M NaCl
    0.02% Tween 20
  3. TE
    10 mM (Tris pH 8.0)
    1 mM EDTA

Acknowledgments

The protocol described here is adapted from one reported previously (Song et al., 2011).

References

  1. Song, C. X., Szulwach, K. E., Fu, Y., Dai, Q., Yi, C., Li, X., Li, Y., Chen, C. H., Zhang, W., Jian, X., Wang, J., Zhang, L., Looney, T. J., Zhang, B., Godley, L. A., Hicks, L. M., Lahn, B. T., Jin, P. and He, C. (2011). Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29(1): 68-72.

简介

5-羟甲基胞嘧啶(5-hmC)是哺乳动物基因组中新发现的DNA修饰。 这个协议是一种高效和选择性的化学方法来标记和捕获5-hmC,利用噬菌体酶,特别是添加葡萄糖部分到5-hmC,这可以反过来用于高通量映射通过下一个 - 生成测序。

关键字:5-羟甲基胞嘧啶, 采集, 测序

材料和试剂

  1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  2. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  3. Trizma碱(Tris)(Sigma-Aldrich,目录号:T1503)
  4. HEPES(GenScript USA,目录号:C01621)
  5. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  6. 二甲基亚砜(DMSO)(Thermo Fisher Scientific,目录号:D128)
  7. Tween-20(Sigma-Aldrich,目录号:P9416)
  8. DBCO-S-S-PEG3 - 生物素结合物(点击化学工具,目录号:A112P3)
  9. 1,4-二硫苏糖醇,超纯(DTT)(Roche Applied Science,目录号:3117006001)
  10. 羟基甲基收集器(Active Motif,目录号:55013)
  11. Wizard基因组DNA纯化试剂盒(Promega Corporation,目录号:A1120)
  12. 10kDa Amicon Ultra-0.5ml离心过滤器(EMD Millipore,目录号:UFC501008)
  13. QIAquick核苷酸去除试剂盒(QIAGEN,目录号:28304)
  14. Micro Bio-Spin 6柱(Bio-Rad Laboratories,目录号:732-6222)
  15. Dynabeads MyOne链霉亲和素C1(Life Technologies,Invitrogen TM,目录号:650-01)
  16. Qiagen MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
  17. UltraPure琼脂糖(Life Technologies,Invitrogen TM,目录号:16500500)
  18. β-葡萄糖基转移酶(β-GT)(New England Biolabs)
  19. 10xβ-GT反应缓冲液(见配方)
  20. 2x结合和洗涤(B& W)缓冲液(参见Recipes)
  21. TE(参见配方)

设备

  1. 声处理装置(Covaris)
  2. 台式离心机
  3. 水浴(Thermo Fisher Scientific)
  4. 凝胶流动装置(Bio-Rad Corporation)
  5. Nanodrop 1000(Thermo Fisher Scientific)
  6. Labquake管振荡器(Barnstead/Thermolyne)
  7. 磁分离架(Promega Corporation,目录号:Z5342)
  8. Qubit 2.0荧光计(Life Technologies,Invitrogen TM)

程序

  1. 根据不同的全基因组测序平台(通常超声到300 bp)将纯化的基因组DNA片段化成短片段。 按照Covaris的手册设置300 bp的片段化程序。 用于超声处理的DNA缓冲液可以是TE(10mM Tris pH 8.0,1mM EDTA),EB(10mM Tris pH8.5)或水。
  2. 使用100-bp DNA标记,验证片段化基因组DNA在2%琼脂糖凝胶上的大小分布。 使用Nanodrop或通过荧光定量测定dsDNA的断裂后的DNA浓度,理想的浓度应高于600ng /μl。
  3. 如果需要,通过10kDa Amicon Ultra-0.5ml离心过滤器或乙醇沉淀来浓缩DNA。 在使用Nanodrop浓缩后再次测量DNA浓度。
  4. 使用适当的起始DNA量,并根据以下条件(脑组织)设置反应
     组件 音量   最终浓度
     水   _μl  
      10xβ-GT反应缓冲液   2μl   1x
     最多10μg基因组DNA   _μl  高达500 ng /μl
      UDP-6-N3-Glc(3mM)   0.67μl   100μM
     β-GT(40μM)   1μl   2μM
     总量   20μl  

  5. 通过吸移混合并在37℃水浴中孵育1小时。
  6. 用QIAquick核苷酸去除试剂盒清除反应,每个柱使用10μgDNA。 用30μl水/柱洗脱并合并。
  7. 将10mg DBCO-S-S-PEG3-生物素缀合物溶解在384μlDMSO中以制备30mM储备溶液并储存在-80℃。 用DMSO稀释储备溶液以制备4.5mM工作溶液并储存在-20℃。 储存溶液在-80℃下稳定数年。 工作溶液在-20°C稳定3个月。
  8. 将来自步骤6)的洗脱的DNA溶液中的DBCO-S-S-PEG3-生物素缀合物工作溶液加至终浓度为150μM(即每30μlDNA溶液中的1μl工作溶液)。
  9. 通过吸移混合并在37℃水浴中孵育1小时。
  10. 用QIAquick核苷酸去除试剂盒清除反应,每个柱使用10μgDNA。用每柱至少30μl水洗脱并合并。理想的组合洗脱体积为100μl,最大体积为150μl。
  11. 使用Nanodrop测量DNA浓度,计算DNA回收率。
  12. 用100μl1x B& W缓冲液按照其手册洗涤50μlDynabeads MyOne链霉亲和素C1三次。用磁力架分离珠子,并将珠子重悬在100μl的2×B& W缓冲液中。
  13. 从步骤11)保存10 ng DNA,将剩余的(100至150μl)加入来自上一步骤的重悬的珠子。
  14. 在室温下孵育15分钟,在Labquake管振荡器上轻轻旋转。
  15. 用磁力架分离珠子,用200μl1×B& W缓冲液洗涤珠子3次。
  16. 通过将珠子在50μl新鲜制备的100mM DTT中温育来洗脱DNA。 在室温下搅拌2小时,在Labquake管摇动器上轻轻旋转。
  17. 用磁性支架分离珠子。 吸出洗脱液并按照其说明将其装载至Micro Bio-Spin 6柱以除去DTT。
  18. 通过Qiagen MinElute PCR纯化试剂盒纯化来自上一步骤的洗脱的DNA,并在10μlEB缓冲液中洗脱DNA。 使用Qubit荧光计定量DNA。 该DNA准备用于下游全基因组测序文库制备。

食谱

  1. 10xβ-GT反应缓冲液
    500mM HEPES(pH 7.9)
    250mM MgCl 2
  2. 2x结合和洗涤(B& W)缓冲液
    10mM Tris(pH7.5) 1mM EDTA
    2 M NaCl
    0.02%Tween 20
  3. TE
    10mM(Tris pH 8.0)
    1mM EDTA

致谢

这里描述的协议改编自先前报道的一个(Song等人,2011)。

参考文献

  1. Song,CX,Szulwach,KE,Fu,Y.,Dai,Q.,Yi,C.,Li,X.,Li,Y.,Chen,CH,Zhang,W.,Jian,X.,Wang,J (2011)的研究结果表明,该方法能有效地解决这一问题。 选择性化学标记显示5-羟甲基胞嘧啶的全基因组分布。 Nat Biotechnol 29(1):68-72。
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  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Szulwach, K. E., Song, C., He, C. and Jin, P. (2012). 5-Hydroxymethylcytosine (5-hmC) Specific Enrichment. Bio-protocol 2(15): e242. DOI: 10.21769/BioProtoc.242.
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